Better temperature uniformity allows for better control of PCR reactions
Effects of Thermal Cycler Well-To-Well Temperature Uniformity on PCR Results
Michelle Cayouette • Jane Moores • Jason Capodanno •
Michael Rahilly
Stratagene
Stratagene’s RoboCycler® temperature cycler #
produces consistent PCR results in every well. The RoboCycler system’s unique
design uses thermal blocks and heating techniques that are optimized for block
temperature uniformity. In this study, fluid temperature profiles and PCR
results were determined using both a RoboCycler ® 96 temperature
cycler and a typical Peltier single-block thermal cycler, the MJ Research
PTC-100. The RoboCycler system showed better fluid temperature well-to-well
uniformity, faster fluid temperature ramping rates, and more consistent
PCR-product banding patterns.
When performing PCR amplifications, reaction components and PCR parameters
are not the only criteria that affect the quality of results; differences may
also exist in the ability of thermal cyclers to maintain a consistent
temperature both within and between experiments. If the well-to-well temperature
across the thermal block is not uniform, it can have a significant impact on the
PCR. This is an especially important consideration for researchers performing
quantitative or diagnostic PCR assays using multiple samples.
The four-block design common to all members of Stratagene’s RoboCycler line
of thermal cyclers provides a higher degree of well-to-well uniformity than the
single-block design of many other thermal cyclers. Each of the three RoboCycler
hot blocks is heated using a microprocessor-controlled resistive element heater,
which covers 95% of the bottom surface of each block. Other thermal cyclers,
such as the MJ Research thermal cyclers, use Peltier thermal electric modules
attached to the bottom of a single block for heating and cooling. However, the
Peltier modules cover only approximately 60% of the bottom surface of the block;
this design creates a temperature gradient from the center to the edges of the
block. Moreover, to ramp temperatures up and down quickly, use of the Peltier
modules requires that the block’s mass be limited.
Maintaining a consistent temperature across a thermal block is easier when
the exposed surface area is kept to a minimum, thereby limiting heat loss due to
convection. Each hot block of the RoboCycler temperature cycler maintains its
programmed temperature throughout the course of a PCR amplification experiment.
A robotic arm moves PCR samples from one preset hot block to another at
user-specified times. This eliminates the need to ramp blocks up and down in
temperature and permits the use of blocks with minimal surface area.
Consequently, RoboCycler temperature cyclers maintain a more consistent
temperature throughout all wells of the block and, hence, provide improved PCR
results.
Fluid Temperature Profiles: RoboCycler 96 vs. MJ Research PTC-100
Figure
1
To compare well-to-well temperature consistency and sample ramping rate,
fluid temperature profiles were determined during thermal cycling using
both a RoboCycler 96 temperature cycler fitted with Stratagene’s
Hot Top Assembly and an MJ Research PTC-100 with heated lid enabled. In
each case, fluid temperature measurements were taken in six tubes positioned
in the four corner wells and two center wells as shown in Figure
1. Similar testing parameters were used for each thermal cycler. T-type
thermocouple temperature probes were inserted into six, 200-µl thin-walled
tubes containing 50 µl of 1X Taq DNA polymerase buffer and
cycled through three cycles of 95ºC for 1 minute, 55ºC for 2 minutes,
and 72ºC for 1 minute. The temperature profile for the MJ Research PTC-100
shows that the sample temperature in tubes in the center of the block
is up to 1.1ºC higher than the sample temperature in tubes positioned
in the edges of the block (Figure
2). The fluid temperature during the denaturation step varied from
95.3 to 96.4ºC, with center wells 1.2 to 1.4ºC higher than the set
temperature. The annealing temperature varied from 54.4 to 54.8ºC, and
the extension temperature varied from 72.4 to 73.2°C. On average, the
PTC-100 changed fluid temperatures at a rate of approximately 0.67°C/second,
with a coefficient of variation (the standard deviation divided by the
mean) of 1.12% between tubes. Therefore, samples placed in different well
positions are not only exposed to different temperatures once ramping
is complete but also change temperature at different rates. Both of these
components combined provide vastly dissimilar PCR conditions between wells
of the Peltier-controlled PTC-100.
Figure
2
The temperature profile of the RoboCycler 96 temperature cycler shows a much
higher degree of well-to-well temperature uniformity (Figure
2). The fluid temperature during the denaturation step only varied
from 95.0 to 95.4°C; the annealing temperature varied from 54.4 to 54.6°C,
and the extension temperature varied from 72.1 to 72.3°C. On average,
the fluid temperature rate of change for the RoboCycler system was approximately
0.85°C/second with a 0.22% coefficient of variation between tubes. This
translates to a 27% increase in the average rate of fluid temperature
change compared to the PTC-100. Also, unlike the PTC-100, the ramping
rates were more consistent from well to well for the RoboCycler system.
These fluid temperature results show that PCR cycling conditions using
the RoboCycler 96 cycler are virtually identical from well to well.
PCR Results: RoboCycler vs. MJ Research PTC-100
Experiments were performed to determine if, and to what extent, the temperature
variability observed across the block of the MJ Research PTC-100 during
fluid temperature testing affected PCR product formation. A fragment of
the trkA gene was PCR-amplified from a cDNA library made from human
brain frontal cortex. A single PCR master mix was prepared large enough
to accommodate six reactions for each of the two thermal cyclers tested.
The 50-µl aliquots from this master mix were placed into 12, 200-µl thin-walled
tubes. Six of these tubes were positioned in a RoboCycler 96 temperature
cycler fitted with Stratagene’s Hot Top Assembly, and six were positioned
in an MJ Research PTC-100 with hot top enabled. As described for the fluid
temperature measurements, all four corners and two center wells were included.
The annealing temperature chosen was known to produce several PCR products
that could be used for comparison. Following thermal cycling, aliquots
from each reaction were run on a vertical 6% polyacrylamide/TBE minigel,
stained with ethidium bromide, and visualized using the Eagle Eye II®
Still Video System (Figure
3).
Figure
3
The yield and specificity of PCR products obtained using the RoboCycler 96
were similar for all wells tested. However, PCR results obtained using the MJ
Research PTC-100 showed a significant degree of well-to-well variability.
Similar results were observed when using the RoboCycler standard paddle and the
MJ Research PTC-100 with the hot top disabled (data not shown). These results
correlate well with the temperature profile obtained from the PTC-100, which
showed a difference of up to 1.1º C from the middle to the edges of the block.
These PCR results corroborate the block temperature profiles and indicate that
researchers can obtain more consistent PCR results from well to well using the
RoboCycler 96 thermal cycler.
Conclusions
The RoboCycler temperature cycler provides more consistent fluid temperature
profiles between center and edge well positions during PCR thermal cycling than
the MJ Research PTC-100. When comparing PCR results from both cyclers, the
RoboCycler 96 also shows a greater well-to-well uniformity of amplified
products. This temperature uniformity results from the RoboCycler system’s
unique four-block design that uses thermal blocks and heating techniques
optimized for block temperature uniformity.
REFERENCES
-
Cayouette, M. et. al. (1997) Strategies 10: 75-76.
* US Patent number 5,525,300 and patents pending